The lymph node (LN) is the primary site of alloimmunity activation and regulation during transplantation. Here, we investigated how fibroblastic reticular cells (FRCs) facilitate the tolerance induced by anti-CD40L in a murine model of heart transplantation. We found that both the absence of LNs and FRC depletion abrogated the effect of anti-CD40L in prolonging murine heart allograft survival. Depletion of FRCs impaired homing of T cells across the high endothelial venules (HEVs) and promoted formation of alloreactive T cells in the LNs in heart-transplanted mice treated with anti-CD40L. Single-cell RNA sequencing of the LNs showed that anti-CD40L promotes a Madcam1+ FRC subset. FRCs also promoted the formation of regulatory T cells (Tregs) in vitro. Nanoparticles (NPs) containing anti-CD40L were selectively delivered to the LNs by coating them with MECA-79, which binds to peripheral node addressin (PNAd) glycoproteins expressed exclusively by HEVs. Treatment with these MECA-79-anti-CD40L-NPs markedly delayed the onset of heart allograft rejection and increased the presence of Tregs. Finally, combined MECA-79-anti-CD40L-NPs and rapamycin treatment resulted in markedly longer allograft survival than soluble anti-CD40L and rapamycin. These data demonstrate that FRCs are critical to facilitating costimulatory blockade. LN-targeted nanodelivery of anti-CD40L could effectively promote heart allograft acceptance.